Nomogram for preoperative estimation of histologic grade in gastrointestinal neuroendocrine tumors.

Background: The treatment strategies and prognosis for gastroenteropancreatic neuroendocrine tumors were associated with tumor grade. Preoperative predictive grading could be of great benefit in the selection of treatment options for patients. However, there is still a lack of effective non-invasive strategies to detect gastrointestinal neuroendocrine tumors (GI-NETs) grading preoperatively. Methods: The data on 147 consecutive GI-NETs patients was retrospectively collected from January 1, 2012, to December 31, 2019. Logistic regression was used to construct a predictive model of gastrointestinal neuroendocrine tumor grading using preoperative laboratory and imaging parameters.The validity of the model was assessed by area under the receiver operating characteristic curve (AUC), calibration curve, and decision curve analysis (DCA). Results: The factors associated with GI-NETs grading were age, tumor size, lymph nodes, neuron-specific enolase (NSE), hemoglobin (HGB) and sex, and two models were constructed by logistic regression for prediction. Combining these 6 factors, the nomogram was constructed for model 1 to distinguish between G3 and G1/2, achieving a good AUC of 0.921 (95% CI: 0.884-0.965), and the sensitivity, specificity, accuracy were 0.9167, 0.8256, 0.8630, respectively. The model 2 was to distinguish between G1 and G2/3, and the variables were age, tumor size, lymph nodes, NSE, with an AUC of 0.847 (95% CI: 0.799-0.915), and the sensitivity, specificity, accuracy were 0.7882, 0.8710, 0.8231, respectively. Two online web servers were established on the basis of the proposed nomogram to facilitate clinical use. Both models showed an excellent calibration curve through 1000 times bootstrapped dataset and the clinical usefulness were confirmed using decision curve analysis. Conclusion: The model served as a valuable non-invasive tool for differentiating between different grades of GI-NETs, personalizing the calculation which can lead to a rational treatment choice.

Pseudoleukocytopenia: a Case Report.

BACKGROUND: Hematology analyzers provide quick and accurate results in most situations. However, spurious results related to the parameters from the complete blood count (CBC) may be observed in several instances. False increases in WBC count can occur for many reasons, including erythroblasts, insufficiently lysed red blood cells (RBC), platelet aggregates, lipids, and cryoglobulins. However, cases of pseudoleukocytopenia due to plasma related factors are rare. METHODS AND RESULTS: Here, we report a case of pseudoleukocytopenia in CBC test. It was identified by the peripheral blood smears and different scatter plots of WNR and WDF channel. The interference from plasma was con-firmed by simulating the state of blood in the WBC channel and plasma exchange. CONCLUSIONS: It was confirmed that the interference of pseudoleukocytopenia was from the plasma. This may be due to the high amount of albumin and other therapeutic drug reactions. Observation of peripheral blood smears and scatter plots can identify this interference.

A novel mutation (Ser951LeufsTer8) in F5 gene leads to hereditary coagulation factor V deficiency.

The current study aims to explore the phenotype and genotype of a novel mutation (Ser951LeufsTer8) of F5 gene combined with polymorphism (R485K) in a family of hereditary coagulation factor V deficiency. The factor V activity and antigen were tested with clotting assay and ELISA. The F5 gene was amplified by PCR with direct sequencing and TA-clone-sequenced. The protein structure and harmfulness of the mutation were studied by Swiss-PdbViewer and bioinformatics software. The prothrombin time and activated partial thromboplastin time of proband were significantly prolonged, factor V activity and factor V antigen both were reduced to less than 20%. Sequencing analysis detected proband with Ser951LeufsTer8 and R485K (Arg513Lys), four family members with novel mutation and their factor V activity and factor V antigen were all decreased about 50%. The Ser951LeufsTer8 is associated with decrease in the factor V level of the family, and it is the first mutation report in the position (Ser951LeufsTer8) with factor V deficiency.

[Phenotypic and genetic analysis of a pedigree affected with hereditary FV deficiency due to a novel deletional variant of F5 gene].

OBJECTIVE: To analyze the phenotype and F5 gene variant in a pedigree affected with hereditary coagulation factor V (FV) deficiency. METHODS: All of the exons, flanking sequences, and 5' and 3' untranslated regions of the F5 gene were subjected to PCR and direct sequencing. Suspected variant sites were confirmed by clone sequencing. Influence of the variants was predicted by using software including ClustalX and Mutation Taster. RESULTS: The prothrombin time (PT) and activated partial thromboplastin time (APTT) of the proband were prolonged to 20.3 s and 59.2 s, respectively, while FV activity (FV:C) and FV antigen (FV:Ag) were reduced by 13% and 17%, respectively. The FV:C and FV:Ag of his father, sister and daughter were decreased to 35%, 37%, 29% and 42%, 46%, 35%, respectively. The proband was found to carry a heterozygous c.2851delT variant in exon 13 of the F5 gene, which caused a frameshift and resulted in a truncated protein (p.Ser923LeufsX8). In addition, a heterozygous c.1538G to A (p.Arg485Lys) variant was found in exon 10. The father, sister and daughter of the proband all carried the p.Ser923LeufsX8 variant, while his mother and son carried the heterozygous p.Arg485Lys polymorphism. His younger brother and wife were of the wild type. Bioinformatic analysis showed that p.Ser923 was highly conserved across various species. Mutation Taster scored 1.00 for the p.Ser923LeufsX8 variant, and the result has predicted a corresponding disease. CONCLUSION: A heterozygous deletional mutation c.2851delT in exon 13 of the F5 gene and a heterozygous c.1538G to A polymorphism harbored by the proband may be associated with the decreased FV level in this pedigree.

[Genetic analysis and clinical features of a pedigree affected with hereditary coagulation factor â ¦ deficiency caused by compound heterozygotic mutations].

OBJECTIVE: To detect potential mutations of the coagulation factor Ⅶ (F7) gene in a pedigree affected with hereditary FⅦ deficiency and explore its molecular pathogenesis. METHODS: The FⅦ antigen (FⅦ:Ag) was analyzed by an enzyme-linked immunosorbent assay (ELISA) method. Prothrombin time (PT), FⅦ activity (FⅦ:C) and other coagulant parameters were quantified with an one-stage clotting assay. The F7 gene was amplified by PCR and sequenced. Mutational sites were confirmed by reverse sequencing. Impact of amino acid substitution was assessed using SIFT and PolyPhen-2 software. Structure of the mutant protein was analyzed using Swiss-pdb Viewer software based on the three-dimensional structure in the Protein Data Bank. RESULTS: The propositus had prolonged PT (36.3 s), with FⅦ:C and FⅦ:Ag significantly reduced to 2% and 44%, respectively. Her father, mother, younger sister and daughter had slightly prolonged PT and reduced FⅦ:C (86%-120%). The FⅦ:Ag of her father and younger sister were also reduced. DNA sequencing revealed that the propositus has carried compound heterozygous mutations (Lys341Glu and IVS6-1G>A) of the F7 gene. Her father and younger sister were heterozygous for the IVS6-1G>A mutation, while her mother and daughter were heterozygous for the Lys341Glu mutation. Bioinformatics analysis indicated that Lys341Glu mutation may affect the stability and function of the FⅦ protein. CONCLUSION: The Lys341Glu and IVS6-1G>A mutations probably underlie the reduced activity of FⅦ in this pedigree.

Phenotypic and genetic analysis of hypofibrinogenemia because of a novel missense mutation in the FGB: Leu121Arg.

: In this study, we found a novel missense gene mutation of fibrinogen (FIB) and it will help us to understand the pathogenesis of this type of disease. The FIB activity (FIB:C) and FIB antigen (FIB:Ag) were detected using matched commercially available kits. To identify the novel missense mutation, the fibrinogen gene sequencing was carried out. Bioinformatics and model analysis were used to study the harm of the mutation. The FIB:C and FIB:Ag of the proband were 0.82 and 1.19 g/l, respectively. Sequencing analysis detected a heterozygous c.425T>G in exon three of FGB gene resulting in p.Leu121Arg. The Leu121Arg mutation was responsible for the decrease of FIB:C, and it was the first report of such a mutation in the world.

[Identification of compound heterozygous mutations of F11 gene in a pedigree affected with heriditary coagulation factor XI deficiency].

OBJECTIVE: To identify potential mutations of F11 gene in a pedigree affected with hereditary coagulation factor XI (FXI) deficiency and explore its molecular pathogenesis. METHODS: Prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB), coagulation factor VIII activity (FVIIIC), coagulation factor IX activity (FIXC), coagulation factor XI activity (FXIC), coagulation factor XII activity (FXIIC) and lupus anticoagulation (LA) of the proband and eight family members were determined. FXI antigen (FXIAg) was determined by enzyme-linked immunosorbent assay (ELISA). For the proband, potential mutations in the exons, flanking introns and 5'-, 3'-untranslated regions of the F11 gene were screened by direct DNA sequencing. The results were confirmed by reverse sequencing. Suspected mutations were detected in other family members. ClustalX-2.1-win and four online bioinformatic tools (PolyPhen-2, PROVEAN, SIFT, and Mutation Taster) were used to study the conservation and possible impact of the mutations. The structure of the mutational sites was processed with Swiss-PdbViewer. RESULTS: The propositus had prolonged APTT (69.6 s), whose FXIC and FXIAg were reduced to 6.0% and 10.7%, respectively. Her mother, elder sister, one younger sister, little brother, daughter and son showed slightly prolonged APTT and moderate FXIC and FXIAg levels. Gene sequencing revealed that the propositus carried a heterozygous nonsense mutation c.738G>A (p.Trp228stop) in exon 7 and a heterozygous mutation c.1556G>C (p.Trp501Ser) in exon 13. Her mother, elder sister and daughter were heterozygous for the p.Trp228stop mutation, while one younger sister and little brother and son were heterozygous for p.Trp501Ser. Her husband and the youngest sister were of the wild type. Phylogenetic analysis suggested that Trp501 was highly conserved among all homologous species. The p.Trp501Ser was predicted to be "probably damaging","deleterious", "affect protein function" and "disease causing" corresponding to PolyPhen-2, PROVEAN, SIFT and Mutation Taster. Model analysis demonstrated that the non-polar Trp501 has two benzene rings, forming a hydrogen bond with Gln512 in the wild type. Once substituted by Ser501, the side chain may form another hydrogen bond with the benzene of His396. This may affect the normal space conformation and stability of FXI protein. CONCLUSION: The compound heterozygous mutations of the F11 gene probably accounted for the low FXI concentration in this pedigree.

Protein C deficiency (a novel mutation: ala291Thr) with systemic lupus erythematosus leads to the deep vein thrombosis.

: The current study aims to explore the phenotype and genotype of a mutation Ala291Thr, which responsible for type I protein C (PC) deficiency in a Chinese woman. The PROC antigen was tested with chromogenic substrate method. PROC gene were amplified by PCR with direct sequencing. Bioinformatics and model analysis were used to study the harm of the mutation. PC activity (PC: A) levels of three members were reduced to 39, 57 and 56%, respectively, PC: antigen was decreased parallelly same as PC: A. Sequencing analysis showed proband with a novel heterozygous c.997G>A point mutation in exon 9 of PROC gene resulting in Ala291Thr. The Ala291Thr mutation is responsible for the decrease of PC: A, which is cross-reacting material negative deficiency and the first reported in the world. This mutation alone may not have significant clinical symptoms, whereas it will cause deep vein thrombosis when combined with systemic lupus erythematosus.

[Phenotypic and mutational analysis of a pedigree affected with hereditary coagulation factor â ¤ deficiency].

OBJECTIVE: To explore the molecular pathogenesis for a pedigree affected with coagulation factor Ⅴ (FⅤ) deficiency. METHODS: Prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB), coagulation factor Ⅱ activity (FⅡ: C), FⅤ activity (FⅤ: C), coagulation factor Ⅶ activity (FⅦ: C), and coagulation factor Ⅹ activity (FⅩ: C) were determined with a STAGO automatic coagulometer. FⅤ antigen (FⅤ: Ag) was detected with enzyme linked immunosorbent assay (ELISA). All exons and their flanking regions, and 5' and 3' untranslated regions of the F5 gene were analyzed by direct sequencing. Suspected mutation was verified by reverse sequencing as well as testing of family members. ClustalX software was used to analyze the conservative property of the mutation sites. PROVEAN and MutationTaster online software was used to predict the effect of the mutation on the protein function. Swiss-pdbViewer was used to analyze the protein model and interaction of amino acids. RESULTS: The PT and APTT of the proband were slightly prolonged to 15.2 s and 41.8 s, respectively. And the FⅤ: C and FⅤ: Ag measured 55% and 62%, respectively. The FⅤ: C and FⅤ: Ag of his father and son were decreased to various extent (60%, 65% and 31%, 40%, respectively). A c.911G>A heterozygous mutation (Gly276Glu) was detected in exon 6 of the proband, for which her father and son were heterozygotes. The same mutation was not found in her mother, brother and husband. Conservation analysis showed that the Gly276 is highly conserved across various species. By bioinformatic analysis, the PROVEAN (scored -6.214) indicated Gly276Glu was harmful, and MutationTaster (scored 0.976) suggested that it is pathogenic. Model analysis suggested there are two hydrogen bonds between Gly276 and Ile298 in the wild type protein. When Gly276 was replaced by Glu276, the original hydrogen bond did not change, but the side chain of Glu was extended, which added steric hindrance with the surrounding amino acids, which resulted in decreased protein stability. CONCLUSION: The heterozygous c.911G>A (Gly276Glu) mutation of the F5 gene probably underlies the decreased level of FⅤin the proband.

A novel mutation (Tyr503Cys) in a severe factor XI deficiency.

: The objective is to study a gene mutation (Tyr503Cys) found in a Chinese consanguineous marriage family with inherited factor XI (FXI) deficiency (cross-reacting material positive, type II). The FXI activity and FXI antigen were tested with clotting assay and ELISA, respectively. The FXI gene was amplified by PCR with direct sequencing. ClustalX-2.1-win and three online bioinformatics softwares were used to study the conservatism and harm of the mutation. The proband had reduced FXI: activity at 13%; three members had decreased to about 35%, all of whom had nomal FXI: antigen. DNA sequencing analysis showed the proband carried a homozygous c.1562A>G point mutation of F11, resulting in Tyr503Cys. Tyr503 was highly conserved among the homologous species. The three bioinformatics softwares indicated that the mutation had affected the function of the protein. The Tyr503Cys mutation was responsible for the decrease of FXI: activity, which is cross-reacting material positive deficiency and the first reported in the world.

Genetic analysis of a novel missense mutation (Gly542Ser) with factor XII deficiency in a Chinese patient of consanguineous marriage.

Coagulation factor XII deficiency is a rare autosomal recessive disorder, which could be found in a consanguineous family. We studied a Chinese family in which the activated partial thromboplastin time (APTT) of the proband had clearly prolonged up to 101.7 s, associated with low FXII activity of 3% and FXII antigen < 1%. To analyze the gene mutation in this FXII-deficient patient, we performed FXII mutation screening, and analyzed the DNA sequence of the F12 gene. A ClustalX-2.1-win and four online bioinformatics software services were used to study the conservatism and effects of the mutation. A transient in vitro expression study was performed to elucidate the possible pathological mechanism. Sequence analysis revealed a homozygous c.1681 G > A point mutation in exon 14, causing a novel Gly542Ser mutation in the catalytic domain. The results of the conservatism and bioinformatics analyses both indicated that the mutation likely affects the function of the protein. Additional expression studies in COS-7 cells showed that the antigen level of mutant FXII (FXII-Gly542Ser) was lower than wild type in culture medium, whereas the corresponding level of FXII antigen in cell lysates was equivalent. These results suggest that the Gly542Ser mutation causes FXII deficiency through intracellular degradation.

Phenotypic and genetic analysis of dysprothrombinemia due to a novel homozygous mutation.

OBJECTIVE: We study the phenotype and genotype of a novel gene mutation of factor II (FII) that leads to dysprothrombinemia, and do the meta-analysis to illuminate its molecular pathogenesis. It will further contribute to our comprehension of the pathogenesis of this type of disease. METHODS: The prothrombin time (PT), activated partial thromboplastin time (APTT) and the activities of other factors were determined by the one-stage clotting method. The prothrombin antigen was measured with enzyme-linked immunosorbent assay (ELISA). Function of the mutant protein was evaluated by thrombin generation tests. Potential mutations in exons, exon-intron boundaries and 5', 3' untranslated sequences of prothrombin gene were screened by polymerase chain reaction and direct sequencing. Suspected mutations were confirmed by reverse sequencing. The structure change of this protein was analyzed by model and bioinformatics analyses. RESULTS: Phenotypic analysis revealed that the proband had an obviously prolonged PT, APTT, reduced prothrombin activity but normal antigen levels. The other tests were normal. Sequencing analysis detected a homozygous g.26329T>G in the catalytic domain resulting in p.Tyr510Asp. His parents and uncle were heterozygous for this mutation. The thrombin generation test showed that the mutant protein had obstacles in thrombin generation. Bioinformatics and model analyses illuminated that the mutation will be probably damaging and perturbing the structure of Na+-binding site, which will affect the activation of prothrombin. CONCLUSION: This was the first report of such a mutation in the position which was associated with dysprothrombinemia.